Analytical liquid chromatography (e.g., high performance liquid chromatography or HPLC) is commonly used in the detection and quantitation of low molecular weight substances in biological samples and body fluid samples (e.g., blood, serum, or plasma). Such samples contain proteins and proteinaceous molecules, which can accumulate on many types of HPLC columns, thereby causing irreversible damage to such columns. For this reason, it is desirable to remove the proteins and other large molecular weight species prior to analysis of the sample for the analyte of interest. Methods of removal of proteins include precipitation, membrane filtration, and liquid-liquid or liquid-solid phase extraction.
An alternate method of removing proteins from a sample is preparative liquid chromatography. In order to adapt such a method to high throughput assays, high flow rates, short elution times, and moderate operating pressures are desirable. Accordingly, porous support materials for use in column chromatography which, simultaneously offer a selective retention of low molecular weight analytes (molecular weight<5000 Dalton), and make possible quantitative separation of proteins and other macromolecular components in a directly injected sample have recently been developed. One example of a column comprising such a porous support is a new column for use in HPLC, known as a monolithic column. Monolithic columns, in contrast to traditional HPLC columns that comprise packed particles, contain a single, solid compound as the stationary phase. This stationary phase is usually made up of a network of polymethacrylate or polystyrene copolymers, or bonded silica forming pores of varying size. Thus, in monolithic columns the mobile phase must flow through the pores of the solid stationary phase. Molecules within the mobile phase are then retained to a greater or lesser extent within the pores of the stationary phase (small molecules diffuse into the pores and are retained while large molecules are excluded from the pores and are not retained). Retention of small molecules may be further enhanced by binding to specific compounds incorporated into the interior of the pores.